WO2007132037A1 - Procédé de synthèse de thymosines - Google Patents

Procédé de synthèse de thymosines Download PDF

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Publication number
WO2007132037A1
WO2007132037A1 PCT/ES2007/000246 ES2007000246W WO2007132037A1 WO 2007132037 A1 WO2007132037 A1 WO 2007132037A1 ES 2007000246 W ES2007000246 W ES 2007000246W WO 2007132037 A1 WO2007132037 A1 WO 2007132037A1
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Prior art keywords
thymosins
thymosin
pharmaceutically acceptable
acceptable salts
resin
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PCT/ES2007/000246
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English (en)
Spanish (es)
Inventor
Jimena Fernandez Carneado
Berta Ponsati Obiols
Javier Clemente Rodriguez
Raimon Rubires Ferrer
Sergi Pavon Fernandez
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Bcn Peptides, S.A.
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Priority to CN200780024724.8A priority Critical patent/CN101484467B/zh
Priority to ES07765833T priority patent/ES2399248T3/es
Priority to US12/300,193 priority patent/US20110166071A1/en
Priority to EP07765833A priority patent/EP2019118B1/fr
Priority to DK07765833.4T priority patent/DK2019118T3/da
Publication of WO2007132037A1 publication Critical patent/WO2007132037A1/fr
Priority to US13/673,871 priority patent/US20130172529A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57581—Thymosin; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/065—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for hydroxy functions, not being part of carboxy functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is within the field of biomedical chemistry.
  • the invention relates to a new method of chemical synthesis of thymosins by synthesis of solid phase peptides with high yield and purity.
  • the procedure is based on the incorporation of pseudoproline derivatives at key positions of the thymosin sequences.
  • the oxazolidine cycle of the pseudoproline prevents the structuring of the growing peptide chain anchored to the resin, preventing the aggregation of the peptidyl resin and the collapse of the synthesis during
  • Thymosins are a family of biochemical and functionally different polypeptides with important physiological functions. First described in 1966 and originally isolated in the thymus, they have subsequently been identified in various tissues and cells [Goldstein, AL, Slat ⁇ r, FD, White, A, Preparation and partial purification of a thymic lymphocytopoietic factor, Proa Nati. Acad. ScL USA 1966, 56 (3), 1010-1017].
  • thymosins that perform different functions, for example oc-thymosins, which have nuclear localization and are involved in DNA replication and / or transcription, and ⁇ -thymosins, which are located in the cytoplasm and show great affinity for G-actin in processes involved in cell motility.
  • the ⁇ - and ⁇ -thymosins play an important role in the regulation of the immune response, vascular biology and pathogenesis of cancer, presenting a great therapeutic potential in addition to its application in diagnosis as molecular markers [Goldstein AL; Badamchian M., Thymosins: chemistry and biological! properties in health and disease, Expert Opin. Biol. Therapy, 4 (4), 559-573].
  • ⁇ -thymosins The subfamily of ⁇ -thymosins is composed of oc-paratymosin, ⁇ -protimosine, ⁇ 1-thymosin and ⁇ 11-thymosin. The most important is the ⁇ 1-thymosin or thymalfasin, originally isolated from thymic tissue, it is a 28 amino acid peptide, acylated in an N-terminal position and with an acid group at its C-terminal end, derived from ⁇ 1-protimosine precursor with 111 amino acids. Due to its immunoregulatory properties, ⁇ 1-thymosin has been used for the treatment of diseases in which the immune system is affected; mainly in hepatitis B, C and liver carcinoma.
  • Chronic hepatitis B is a disease related to a dysfunction of the immune system, it carries a high risk of suffering from cirrhosis and hepatocellular carcinoma.
  • ⁇ 1-Thymosin acts as a modulator of the immune system by activating the production of NK cells in various animal and human models and increasing the production of CD3, CD4 and CD8 cells in patients with chronic hepatitis B and cancer. It also activates the production of Th1 cytokines, related to a strong antiviral immune response.
  • ⁇ 1-Thymosin increases MHC class 1 expression levels in cell cultures, maintaining the ability of the immune system to respond to infectious agents [Herzenberg, LA .; De Rosa, SC; Dubs, JG, Glutathione deficiency is associated with impaired sun / ival in HIV disease, Proc. Nati Acad. Sci. USA, 1997, 94, 1967-1972]
  • mice infected with various pathogens Listeria monocytogenes, Candida albicans, Pseudomonas aeruginosa and Serratia marcescens
  • mice immunocompromised with 5-fluorouracil [Ishitsuka, H; Umeda, Y. Nakamura, J. Yagi, Y. Protective activity of thymosin against opportunistic infections in animal models. Immunol cancer. Immunother 1983, 14, 145-150].
  • oc1-thymosin increases the survival of adult or immunosuppressed animals infected with herpes simplex virus, influenza virus or Candida albicans
  • herpes simplex virus influenza virus or Candida albicans
  • RP Casillas
  • RL The effect of thymosin alpha 1 on immunity to influenza in Aged mice Aging: immunology and infectious. disease Yol 1., NewYork, NY: Mary Ann Liebert, INC; 1988; 31-40; Bistoni , F., Marconi, P., Frati, L, Bonmassar, E, Garaci, E. Increase of mouse resistance to candida albicans infection by thymosin alphai. Infect Immun. 1982, 36, 609-614].
  • the synergistic effect of oc1-thymosin with other cytokines in the treatment of hepatitis C has been revealed in trials with mice infected with influenza A virus treated with a combination of ⁇ 1-thymosin, interferon IFN ⁇ / ⁇ and the antiviral amantadine.
  • the combination of the three compounds substantially improves the survival rate [D'Agostini C, Palamara, AT, Favalli, C. Efficacy of combination therapy with amantadme, thymosin alpha 1 and alpha / beta interferon in mice infected with influenza A virus. Int. J. Immunopharmacol. 1996, 16, 95-102].
  • oc-thymosin has demonstrated its efficacy in the prevention of intestinal cancer [Moody, TW, Leyton, J., Zia, F, Tuthill, C, Badamchian, M. Goldst ⁇ in, AL, Thymosin alphai is chemopreventive for lung adenoma formation in A / J mice, Cancer Lett. 2000, 155, 121-127], breast cancer, reducing the incidence of cancer development as a result of the stimulation of the immune system in rats [Moody, TW, Tuthill, C, Badamchian, M., Goldstein, A.
  • Thymosin alphai inhibits mammary carcinogenesis in Fisherrats, Peptides, 2002, 23 (5), 1011-1014Jy glioblastoma
  • Patent WO03049697, Thymosin-alphai is used as an -adjuvant in combination with carmustine (BCNU) as an effective treatment- for malignant glioblastoma, Wands, JR De la Monte S., Rhode Island Hospital (US), 06-30-2005].
  • mice The combined therapy of interferon IFN ⁇ -2b, ⁇ 1-thymosin and cyclophosp amide has given good results, reducing the size of melanoma in mice [Pica, F., Frasc ⁇ etti, M., Matteucc ⁇ , C, Tuthill, C, Ras ⁇ , G ., High doses of thymosin alpha 1 enhance the anti-tumor efficacy of combination chemo-immunotherapy for murine B16 melanoma, Anticancer Res. 1998, 18, 3571-3578].
  • thymosin fraction 5 preparations des- (25-28) - ⁇ 1-thymosin and ⁇ 11-thymosin
  • Thimosin ⁇ 11 a peptide related to thymosin al isolated from calf thymosin fraction 5, Calderella, J., Goodall, GJ. , Felix, A.M., Heimer, E.P., SaMn, S.B., Horecker, B.L, Proc. Nati Acad. Sd. USA, 80, 7424-742].
  • the second subfamily of thymosins are the ⁇ -thymosins that constitute a family of proteins ( ⁇ -4, ⁇ -8, ⁇ -9, ⁇ -10, ⁇ -11, ⁇ -12, ⁇ -15 thymosins) with high homology in its peptide sequence (see Table 1).
  • Table 1 shows the sequences of the thymosins.
  • ⁇ 4-Thmosin contains 43 amino acids and is expressed in stages of embryonic development corresponding to the development of the heart. This discovery, carried out in preclinical animal studies, has highlighted the potential of ⁇ 4-thymosin for the treatment of patients who have suffered a myocardial infarction. In preclinical studies with mice to which heart attacks were induced, treatment with ⁇ 4-thymosin protein promoted the differentiation of endothelial cells and stimulated the migration of cardiac cells, improving the survival capacity of said cells [Bock-Marquette, /., Saxena, A., White, MD, DiMaio, JM. , Srivastava, D., Thymosin ⁇ 4 activates integrin-linked kinase and promote cardiac cell migration, survival and cardiac repair, Nature, 2004, 432, 466-472]
  • ⁇ 4-Thymosin also regulates a series of inflammatory cytokines and chemokines, controls the function of the actin protein, binding to it, and induces the production of Iminma-5, a protein necessary for the correct adhesion of some types of mammalian cells and an important component of the wound repair process, thus facilitating reepithelialization and inducing collagen deposition [Bubb, MR, Thymosin beta 4 interactions, Vitam. Horm 2003, 66, 297-316].
  • ⁇ 4-thymosin include increased hair growth by activating hair follicle stem cells, called “hair follicle stem cells” [WO / 063775 A2, Methods and compositions for the promotion of hairgrowth ⁇ tilizing actin binding peptides] .
  • hair follicle stem cells called “hair follicle stem cells” [WO / 063775 A2
  • Methods and compositions for the promotion of hairgrowth ⁇ tilizing actin binding peptides] are also for the treatment of various types of chronic dermatological indications such as chronic chronic venous stasis ulcers, chronic pressure ulcers, bullous epidermolysis, a group of inherited disorders characterized by the formation of blisters in response to trauma mechanical and treatment of corneal wounds.
  • LKKTETQ fragment of ⁇ 4-thymosin facilitates the healing of dermal wounds in adult animals [Philp D., Huff, D., Song Gho, Y .; Hannappel, E, Kleinman, T., The actin binding site on thymosin ⁇ 4 promotes angiogenesis, FASEBJ., 17, 2103-2105].
  • ⁇ 15-Thymosin is a 44 amino acid peptide overexpressed in prostate cancer, where it favors the spread of the tumor outside the prostate gland.
  • This peptide is being developed as a biomarker for the diagnosis of said cancer, for the discrimination between benign and malignant prostatic tumors and for proper treatment [Hutchinson, LM., Chang, EL, Becker, CM., Ushiyama, N., Behonick, D., Shih, MC, DeWoIf, WC, Gastón, SM, Zetter, BR, Development of a sensitive and specific enzyme-linked immunoassay for thymosin beta 15, a urinary blomarker of human prostate cancer, Clin. Biochem 2005, 38 (6), 558-571].
  • ⁇ 15-Thymosin has also been patented for use in wound healing and regeneration and can be applied for this indication in the near future [Patent CN1579539, Thymic hormone beta 15 to promote wound healing and generation of block, Gan Chunyu (CN) J .
  • ⁇ 10-imosin is made up of 43 amino acids and is overexpressed in embryonic and tumor cells such as colon adenocarcinomas / torture, E., Tomita, Y., Brown, LF, Kocher, O., Dvorak, HF, Differential expression of thymosin ⁇ -10 by early passage and senescent vascular endothelium is modulated by VPF / VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis, FASÉB J., 2 ⁇ 1, 15,458-466]
  • ⁇ 10-Thymosin has a great homology in Ia ⁇ 4- Thymosin since both bind to actin and inhibit its polymerization [Yu, FX, Lin, SC, Morrison-Bogorad, M., Atkinson, MA, Yin, HL, Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins , J.
  • ⁇ 8-thymosin contains 39 amino acids and a high degree of homology (79%) with ⁇ 4-thymosin.
  • the ⁇ -thymosin has 41 amino acids and differs from the ⁇ 9-thymosin in an additional dipeptide in the C-terminal region, presenting an 82% homology with the ⁇ 4-thymosin.
  • the ⁇ 11-thymosin with 42 amino acids has a 78% homology with respect to the ⁇ 4-thymosin.
  • the ⁇ 12-thymosin with 43 amino acids is 79% homologous to the ⁇ 4-thymosin and 84% to the ⁇ 11-thymosin (see Table 1).
  • the first patented synthetic thymosin procedure was to obtain ⁇ 1-thymosin.
  • This procedure proposed the convergent synthesis in solution of two fragments of fifteen (15) and thirteen amino acids (13), which had previously been synthesized in solid phase with the Boc / Bzl strategy [US4148788, Synthesis of thymosin alpha 1, Hoffmann La Roche , 1979].
  • This procedure was subsequently implemented in a strategy carried out in solution in which seven (7) fragments were used [Patent US4504415, Intermediates for thymosin alpha 1 and desacetyl thymosin alpha 1, Hoffmann La Roche, 1985],
  • a variant of the ⁇ 4-thymosin in which the Ser15, Ala40 and Ser41 positions of the ⁇ 4-thymosin are replaced by Ala, Thr and Ser respectively, is the ⁇ 4 Xen- thymosin that has already been synthesized according to a linear Fmoc / tBu strategy , and from which a crude purity of 48-53% has been obtained [Voelter, W., Kaiser, T., Hannapel, E., Echner, H, Synthesis of thymosin ⁇ 4 Xen and its comparison with the natural tritetraconpeptide, J.Prakt.Chem., 1999, 341, 1, 47-51].
  • ⁇ 15-thymosin is obtained by a similar strategy, that is, a linear synthesis based on the Fmoc / tBu strategy.
  • This synthesis leads to the product ⁇ 15-thymosin, but also involves long and incomplete couplings, which include an acetylation step, reducing the purity of the crude oil obtained [Koutrafour ⁇ , V., Leondiadis, L 1 Ferder ⁇ gos, N., Avgoustakis, K., Livaniou, E., Evangelatos, GP, Ithakissios, DS Synthesis and angiogenetic activity in the chick chor ⁇ oallantoic membrane model of thymosin beta-15, Peptides, 2003, 24, 107-115].
  • the overall yields obtained are not low (40% of ⁇ 10-thymosin) and (45% of ⁇ 15-thymosin) although the synthesis procedure in anticipation of large-scale production needs to be optimized.
  • the hydrophobic interactions of the growing protected peptide chain give rise to low coupling yields, incomplete deprotections and the obtention of secondary products.
  • the use of pseudoprolines recently introduced in synthesis of long peptides is a substantial improvement for the state of the art in peptides with a great tendency to structure during the elongation of the chain.
  • Pseudoproiins are cyclic derivatives of Ser, Thr or Cys that serve as protective groups of these amino acids (Formula I) and facilitate the synthesis of difficult sequences, interrupting the structuring of the growing peptide chain still anchored to the resin and preventing the aggregation of Ia peptidyl-resin and the collapse of the synthesis
  • Table 3 shows the sequences of the thymosins; Ser and Thr that could be replaced by pseudoprolines are underlined.
  • the present invention describes a new synthetic process for the preparation and industrial production of thymosins ( ⁇ and ⁇ ) using protected pseudoproline dipeptides in strategic positions (See Table 4).
  • the thymosin synthesis method proposed in the present invention describes a procedure consisting of the following steps: a) Linear synthesis of solid phase peptides on resin applying the Fmoc / tBu methodology, b) Use of X-Thr or X-Ser, where X is any amino acid, in the form of pseudoproline dipeptides in strategic positions of the sequence according to the following guidelines: b.1) The insertion of pseudoproline dipeptides is not necessary in the C-terminal position or in the N-terminal , b.2) Incorporation of at least one pseudoproline dipeptide in the sequence, b.3) The structure destabilizing effect of the pseudoprolines maintains its influence on the following 5-6 amino acids, b.4) The optimal separation between pseudoproline dipeptides is of 5-6 amino acids.
  • Table 3 shows those sequences of the thymosins with all possible positions for the incorporation of pseudoproline dipeptides.
  • the object of the present invention was developed because the synthesis of long peptides is a challenge due mainly to poorly efficient couplings and deprotections, derived from the aggregation effects also found in sequences that are not very long but especially hydrophobic or repetitive.
  • the present invention aims to solve this technical problem and it proposes a synthetic process for obtenciórr industr-ia ⁇ thymosins "based eo the synthesis of peptides in solid phase, applying the methodology Fmoc / tBu upon a type 2-chlorotrityl resin incorporating X-Ser and X-Thr in key positions as pseudoproline dipeptides.
  • the " solid phase of fas " synthesis with a C-termine acid function is carried out from a 2-chlorotritic chloride resin [Barios K., Chatzi, O., Cats, D., Stavropoulos, G , 2-Chlorotr ⁇ tyl chloride resin, Int. J. Peptide Protein Res., 1991 (37), 513-520].
  • This resin facilitates the incorporation of the first amino acid and minimizes the secondary reaction of racemization.
  • This resin also reduces the risk of diketopiperacin formation in the basic treatment with 20% piperidine in DMF after the incorporation of the second amino acid on the resin.
  • the present invention is characterized in that it incorporates Thr or Ser amino acids, according to the peptide sequence, through or by adding pseudoproline dipeptides at strategic positions of the peptide sequence.
  • the present invention is therefore characterized in that it incorporates Thr or Ser amino acids, according to the peptide sequence, by adding pseudoproline dipeptides at strategic positions of the peptide sequence, following the following guidelines:
  • pseudoproline dipeptides a) The insertion of pseudoproline dipeptides is not necessary in the C-terminal position or in the N-terminal, b) The incorporation of at least one pseudoproline dipeptide in the sequence, c) The structure destabilizing effect of the pseudoprolines maintains its influence in the following 5-6 amino acids, d) The optimal separation between pseudoproline dipeptides is 5-6 amino acids. e) The minimum separation between two pseudoproline dipeptides or a pseudoproline and a proline is preferably of two residues, f) The synthetic result is optimal if the pseudoproline dipeptide is in a region of hydrophobic residues. Obtaining and optimizing a synthetic process for the production of thymosins at the industrial level has been a great effort. Our laboratory has addressed other synthetic strategies to obtain thymosine without success.- To obtain a profitable productive process for the production of ⁇ 4-thymosin, three different strategies have been studied:
  • Second strategy through solid phase linear synthesis A second approach, the step-by-step solid phase linear synthesis of ⁇ 4-thymosin gave rise to a low purity synthesis crude, unfeasible for an industrial process of producing ⁇ 4-thymosin given the difficulty of the purification stage and the low overall performance obtained.
  • the third strategy has involved the resolution of a problem, the obtaining of thymosins according to a competitive process on an industrial scale.
  • the use of pseudoprolines has been incorporated in the solid phase linear synthesis with the Fmoc / tBu strategy.
  • pseudoprolines are non-natural amino acids derived from Cys, Ser or Thr with a structure similar to proline.
  • the cyclic oxazolidine ring prevents the structuring of the growing peptide chain still anchored to the resin and blocks the aggregation of the peptidyl resin and the collapse of the synthesis.
  • the final acid treatment of peptidyl resin regenerates Ser o. Thr of. the native sequence . .
  • the peptidyl resin is subjected to a final treatment with trifluoroacetic acid and carbocation captors to cleave the peptide from the resin and Eliminate the protective groups of the side chains and the pseudoproline protection.
  • the resulting crude is purified by RP-HPLC and the set of pure fractions are combined and lyophilized, obtaining the final products with a purity of more than 99%, with an overall yield greater than 50%.
  • Figure 1 shows the crude of ⁇ 4-thymosin isolated according to strategy 2 and strategy 3.
  • Table 2 (exposed in previous lines) we can observe the difference in yields in these strategies and others previously published for the synthesis of ⁇ - thyroidins.
  • Table 2 (exposed in previous lines) we can observe the difference in yields in these strategies and others previously published for the synthesis of ⁇ - thyroidins.
  • Table 2 (exposed in previous lines) we can observe the difference in yields in these strategies and others previously published for the synthesis of ⁇ - thyroidins.
  • Table 2 The approximation of linear synthesis in solid phase following an Fmoc / tBu strategy without the use of pseudoprolines results in a crude oil difficult to purify (Table 2).
  • Figure 1A shows the use of pseudoproline dipeptides in a linear Fmoc / tBu strategy significantly improves the performance and purity of the " final product.
  • the invention is based on the use of pseudoproline dipeptides in key positions of the synthesis, following the aforementioned guidelines, avoiding sequences that cause the aggregation of the growing chain on the resin and thus eliminating problems of incomplete coupling and deprotection that lead to a peptide crude of low quality and therefore at a low synthesis yield, with the consequent difficulty in the purification process.
  • DlPCDl Diisopropylcarbodiimide.
  • DMF N, N-dimethylformamide.
  • Fmoc 9-FIuoroenümethoxycarbonyl.
  • HPLC High Pressure Liquid Chromatography. ⁇ L: microliters. ⁇ mol: micromoles. tBu: Tere-butyl.
  • TFA Trifluoroacetic acid
  • the present invention relates to a process for obtaining thimosines and / or their pharmaceutically acceptable salts by solid phase synthesis on polymeric supports comprising the following steps:
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts in which ⁇ -thymosins and / or their pharmaceutically acceptable salts are obtained.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts in which ⁇ -thymosins and / or their pharmaceutically acceptable salts are obtained.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts in which the synthesis is carried out on polymeric supports.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts where in step a) a chloro-trityl type resin is used.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts where in step a) a pMBHA type resin is used.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts where in step b) the incorporation of Ser or Thr is carried out additionally as pseudoproline and / or Ser or Thr modified dipeptides in Pseudoproline form in the peptidyl resin.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts where preferably the minimum separation between two pseudoproline dipeptides or a pseudoproline and a proline is two amino acids.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts where preferably the pseudoproline dipeptide is added in a region of hydrophobic amino acids.
  • the present invention relates to a process for obtaining thymosins and / or their pharmaceutically acceptable salts in which if after the step d) the amino acid Met appears in the sequence, a treatment of crude disterbutylation with AcOH is also performed.
  • the present invention relates to an isolated and / or purified compound obtained by the above procedure comprising an amino acid sequence Ia which has at least 80% identity with the sequences SEQ.ID.N.1 to SEQ.ID.N.11 over its entire length.
  • the present invention relates to an isolated and / or purified compound obtained by the above procedure consisting of an amino acid sequence chosen from the group formed by the sequences SEQ.iD.N.1 to SEQ.ID.N.11, by their sequences complementary and / or by their homologous sequences and / or by their equivalent functional sequences.
  • the present invention relates to an isolated and / or purified compound obtained by the above procedure that includes fragments of at least one amino acid sequence chosen from the group formed by the sequences SEQ.ID.N.1 to SEQ.ID.N.11 , for its complementary sequences, for its homologous sequences and for its equivalent functional sequences.
  • the present invention relates to an isolated and / or purified compound obtained by the above procedure in which any of the amino acids comprised in the sequences SEQ.ID.N.1 to SEQ. ID. N.11 is selected from the group consisting of L-amino acids, D-amino acids, N-methyl amino acids, unnatural amino acids, Met-tBu or Met-oxidized.
  • the present invention relates to an isolated and / or purified compound obtained by the above procedure that includes modifications to the N-terminal amino acid selected from acylations and / or pegylations.
  • the present invention refers to the use of the compound obtained by the process mentioned above in the preparation of a medicament.
  • the present invention relates to the use of the compound obtained by the aforementioned procedure in the preparation of a medicament for the regulation of the immune response, vascular biology and pathogenesis of cancer.
  • the incorporation of the C-terminal residue Fmoc-Asn-OH on the chlorotrityl resin 2 is carried out with an amino acid defect, in order to obtain a functionalization of 0.85 mmol / g after the incorporation of the first amino acid.
  • resin 1.6 mmol / g of resin, 4 mmol
  • amino acid 2.13 mmol, 0.5 eq.
  • the resin and the amino acid are weighed in separate containers and allowed to dry under vacuum for a minimum of 4 hours.
  • the amino acid is dissolved in 25 mL of dry DCM (on 4A sieve).
  • DIEA (0.7 mL, 1.6 mmol, 1 eq.) Is added and stirred for 5 min.
  • the incorporation of the following amino acids is carried out in all cases with an excess of 2.5 eq of amino acid, HOBT and DIPCDl with respect to the functionalization of the resin after the incorporation of the first amino acid (0.85 mmol / g). It is allowed to react for 40-60 min and subsequently the deprotection of the Fmoc group is carried out with 20% piperidine in DMF for 5 min 2 times and for 10 min another 2 times.
  • the Fmoc-Ile-Thr dipeptide ( ⁇ Me - Me pro) -OH (corresponding to amino acids 11-12 of the sequence of ⁇ 1-thymosin) was incorporated using 2.5 eq. of dipeptide, HOBT and DIPCDI, allowing it to react for 1 h. Coupling efficiency is controlled by the ninhydrin test. If it is positive, it is reactivated using 1/3 eq. HOBT and DIPCDI and if after reactivation it is still positive, it is re-coupled using Vz eq. of Fmoc-AA-OH, HOBT and DIPCDI with respect to the equivalents used in the first coupling. If after reactivating, re-couple and reactivate again The ninhydrin test is still positive, acetylation is performed using 2.5 eq. Ac 2 O, and DIEA for 15 min.
  • the yield at the end of the synthesis is quantitative in obtaining Ac-Ser (tBu) - Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Ser (tBu) -Glu (OtBu) -lle-Thr ( ⁇ Me - Me pro) - Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Lys (Boc) -QIu (OtBu) -Val- Val-Glu (OtBu) -G!
  • ⁇ 4-Thymosin was synthesized following a solid phase peptide synthesis strategy using Fmoc / tBu protectors.
  • the amino acid N ⁇ -Fmoc-protected 0.5 eq
  • DIEA 3 eq.
  • the resin is filtered in a synthesis reactor equipped with a filter plate and a key and the following were washed with DCM and DMF.
  • the resin is treated with 5% piperidine in DMF 1 x 10 min and 20% piperidine in DMF 1 x 15 min and rinsed with DMF.
  • the remaining amino acids were incorporated using N ⁇ -Fmoc-protected (3 eq.), HOBT (3 eq.) And DIPCDI (3 eq.) In DMF, equivalents calculated with respect to the functionalization of the resin once the first amino acid is incorporated.
  • the pseudoproline dipeptides were incorporated in the synthesis as Fmoc-Lys (Boc) -Ser ( ⁇ Me, Me pro) -OH instead of amino acids 14-15 and Fmoc- Glu (OtBu) -Thr ( ⁇ Me, Me pro) -OH in amino acid positions 21-22 and 32-33. Said positions were chosen among those possible because of their disposition in the peptide sequence, optimal proximity to the C-terminal position, optimal proximity to Pro residues and optimal distance between pseudoprolines.
  • Coupling efficiency is controlled with the ninhydrin test. If it is positive, it is reactivated using V & eq. KQBT and DIPCD1 and if after -activation is still positive ⁇ it is re-coupled using Vz eq. From Fmoc-AA-OH, HOBT and DIPCDI with respect to the first coupling. If after reactivating, re-coupling and reactivating again, the ninhydrin test is still positive, acetylation is carried out using 2.5 eq. Ac 2 O, and DIEA for 15 min; - - - - - - - -
  • the synthetic procedure was similar to that used for ⁇ 4-thymosin.
  • the dipeptide Fmoc- Lys (Boc) -Ser ( ⁇ Me> M ⁇ pro) -OH was used in substitution of amino acids 14-15, Fmoc- Asn (Trt) -Thr ( ⁇ Me ' Me pro) -OH in the positions of amino acids 21-22 and Fmoc-Glu (OtBu) - Thr ( ⁇ Me ' Me pro) -OH at positions 32-33. Only one re-coupling was necessary in Ia Lys (3). . . . . . . . . . . ... . . . . . .
  • the peptide was cleaved from the resin by reacting it with a mixture of TFA: H 2 O 95: 5 for 1 h 30 min, precipitated with diethyl ether and lyophilized.
  • the crude was purified on a semi-preparative RP-HPLC and the final product was characterized by mass spectrometry in an ESI-MS equipment. Purity: 98% Yield: 50%

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Abstract

L'invention concerne un procédé pour l'obtention de thymosines et/ou de leurs sels pharmaceutiquement acceptables par synthèse en phase solide sur supports polymères. Ce procédé consiste à réaliser la synthèse linéaire de peptides dérivés du thymus en phase solide, à incorporer au moins un Thr ou Ser sous forme de dipeptide de pseudo-proline dans la séquence, à obtenir les thymosines par traitement d'une peptidyl-résine avec de l'acide trifluoroacétique, et à purifier les thymosines par chromatographie RP-HPLC. La présente invention concerne également le composé isolé et/ou purifié obtenu au moyen de ce procédé ainsi que l'utilisation dudit composé dans la préparation d'un médicament.
PCT/ES2007/000246 2006-05-10 2007-04-25 Procédé de synthèse de thymosines WO2007132037A1 (fr)

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CN200780024724.8A CN101484467B (zh) 2006-05-10 2007-04-25 合成胸腺素的方法
ES07765833T ES2399248T3 (es) 2006-05-10 2007-04-25 Procedimiento para sintetizar timosinas
US12/300,193 US20110166071A1 (en) 2006-05-10 2007-04-25 Method for synthesizing thymosins
EP07765833A EP2019118B1 (fr) 2006-05-10 2007-04-25 Procédé de synthèse de thymosines
DK07765833.4T DK2019118T3 (da) 2006-05-10 2007-04-25 Fremgangsmåde til syntetisering af thymosiner
US13/673,871 US20130172529A1 (en) 2006-05-10 2012-11-09 Method for synthesizing thymosins

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ES200601197A ES2288118B1 (es) 2006-05-10 2006-05-10 Procedimiento para sintetizar timosinas.

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148788A (en) 1978-01-23 1979-04-10 Hoffmann-La Roche Inc. Synthesis of thymosin α1
US4504415A (en) 1983-04-04 1985-03-12 Hoffman-La Roche Inc. Synthesis of thymosin α1 and desacetyl thymosin α1
ES2103966T3 (es) * 1991-09-13 1997-10-01 Alpha 1 Biomedicals Inc Uso de una timosina en el tratamiento de hepatitis c.
ES2119581A1 (es) * 1992-08-05 1998-10-01 Orpegen Med Molekularbioforsch Utilizacion de fragmentos de timosina y/o sus derivados
US5856440A (en) 1988-05-10 1999-01-05 Alpha-1 Biomedicals, Inc. Solid phase process for synthesizing thymosin α1
WO2003049697A2 (fr) 2001-12-10 2003-06-19 Rhode Island Hospital Traitement du glioblastome par la thymosine alpha 1
WO2003063775A2 (fr) 2002-01-25 2003-08-07 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Procedes et compositions favorisant la pousse des cheveux a l'aide de peptides de liaison d'actine
ES2192839T3 (es) * 1998-03-28 2003-10-16 Univ Glasgow Timosina beta 4 oxidada.
CN1579539A (zh) 2003-07-25 2005-02-16 干春玉 胸腺素β15促进伤口愈合及血管生成

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60152243A (ja) * 1984-01-17 1985-08-10 Mitsubishi Electric Corp 分割固定子の電機子巻線
US5190918A (en) * 1990-02-22 1993-03-02 W. R. Grace & Co.-Conn. Peptide fragments and analogs of thrombospondin and methods of use
CN1042493C (zh) * 1993-01-19 1999-03-17 祁岩超 人胎胸腺素的制备方法
CN1058500C (zh) * 1993-02-03 2000-11-15 施塞克龙药品公司 胸腺素α－1衍生物
US6288033B1 (en) * 1998-09-25 2001-09-11 Sciclone Pharmaceuticals, Inc. Treatment of hepatitis B infection with thymosin alpha 1 in combination with lamivudine or in combination with lamivudine and famciclovir
EP1891104B1 (fr) * 2005-05-04 2009-08-26 Lonza Ag Thymosine alpha1 liee en phase solide et sa synthese

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148788A (en) 1978-01-23 1979-04-10 Hoffmann-La Roche Inc. Synthesis of thymosin α1
US4504415A (en) 1983-04-04 1985-03-12 Hoffman-La Roche Inc. Synthesis of thymosin α1 and desacetyl thymosin α1
US5856440A (en) 1988-05-10 1999-01-05 Alpha-1 Biomedicals, Inc. Solid phase process for synthesizing thymosin α1
ES2103966T3 (es) * 1991-09-13 1997-10-01 Alpha 1 Biomedicals Inc Uso de una timosina en el tratamiento de hepatitis c.
ES2119581A1 (es) * 1992-08-05 1998-10-01 Orpegen Med Molekularbioforsch Utilizacion de fragmentos de timosina y/o sus derivados
ES2192839T3 (es) * 1998-03-28 2003-10-16 Univ Glasgow Timosina beta 4 oxidada.
WO2003049697A2 (fr) 2001-12-10 2003-06-19 Rhode Island Hospital Traitement du glioblastome par la thymosine alpha 1
WO2003063775A2 (fr) 2002-01-25 2003-08-07 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Procedes et compositions favorisant la pousse des cheveux a l'aide de peptides de liaison d'actine
CN1579539A (zh) 2003-07-25 2005-02-16 干春玉 胸腺素β15促进伤口愈合及血管生成

Non-Patent Citations (43)

* Cited by examiner, † Cited by third party
Title
ANDREONE, P.; CURSARO, C.; GRAMENZI, A: "A randomized controlled trial of thymosin alpha 1 versus interferon alpha treatment in patients with hepatitis B antigen antibody and hepatitis B virus DNA, positive chronic hepatitis B", HEPATOLOGY, vol. 24, 1996, pages 774 - 777J
BARLOS K. ET AL.: "2-Chlorotrityl chloride resin", INT. J. PEPTIDE PROTEIN RES., vol. 37, 1991, pages 513 - 520J
BARLOS K.; CHATZI, O.; GATOS, D.: "Stavropoulos, G, 2-Chlorotrityl chloride resin", INT. J. PEPTIDE PROTEIN RES., vol. 37, 1991, pages 513 - 520J
BISTONI, F. ET AL.: "Increase of mouse resistance to candida albicans infection by thymosin alpha1", INFECT. IMMUN., vol. 36, 1982, pages 609 - 614J
BOCK-MARQUETTE, I. ET AL.: "Thymosin /34 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair", NATURE, vol. 432, 2004, pages 466 - 472, XP002358431, DOI: doi:10.1038/nature03000
BUBB, M.R.: "Thymosin beta 4 interactions", VITAM. HORM., vol. 66, 2003, pages 297 - 316J
CALDERELLA, J. ET AL.: "Thymosin a11: a peptide related to thymosin a1 isolated from calf thymosin fraction 5", PROC. NATL. ACAD. SCI. USA, vol. 80, pages 7424 - 742
COLLEDGE, D. ET AL.: "Antiviral effects of thymosin alpha 1 versus leukocytic interferon in primary cultures of duck hepatocytes persistently infected with duck HBV in vitro", SECOND INTERNATIONAL CONFERENCE ON THERAPIES FOR VIRAL HEPATITIS, 1997
D'AGOSTINI C.; PALAMARA, A.T; FAVALLI, C.: "Efficacy of combination therapy with amantadine, thymosin alpha 1 and alpha/beta interferon in mice infected with influenza A virus", INT. J. IMMUNOPHARMACOL., vol. 16, 1996, pages 95 - 102, XP009062682, DOI: doi:10.1016/0192-0561(96)00002-1
ECHNER, H.; VOELTER, W.: "A new synthesis of thymosin a1", LIEBIGS ANNALEN DER CHIMIE, vol. 11, 1988, pages 1095 - 1097
ECHNER, H.; VOELTER, W: "A new synthesis of thymosin a1", LIEBIGS ANNALEN DER CHIMIE, vol. 11, 1988, pages 1095 - 1097J
EFFROS, R.B.; CASILLAS, A.; WADLFORD, R.L.: "The effect of thymosin alpha 1 on immunity to influenza in aged mice. Aging: immunology and infectious disease", vol. 1, 1988, MARY ANN LIEBERT, INC, pages: 31 - 40
FELIX, AM. ET AL.: "Synthesis of thymosin a1 by fragment condensation using tert-butyl side chain protection", INT. J. PEPT. PROTO RES., vol. 26, no. 2, 1985, pages 130 - 148
GARACI, E.; MILANESE, G.; VELLA, S.: "A randomized controlled study for the evaluation of the activity of a triple combination of zidovudine, thymosin alpha 1 and interferon alpha in HIV-infected individuals with CD4 counts between 200 and 500 cells/mm3", ANTIVIRAL THERAPY, vol. 3, 1998, pages 103 - 11
GERIN, J.L. ET AL.: "Innovations in antiviral development and the detection of virus infection", 1992, JEFFERSON MEDICAL, article "A preliminary report of a controlled study of thymosin alpha 1 in the woodchuck model of hepanavirus infection", pages: 121 - 123
GIULIANI, C.; NAPOLITANO, G.; MASTINO, A: "Thymosin alpha-1 regulates MHC class / expression in FRTL-5 cells at transcriptional level", EUR. J. IMMUNOL., vol. 30, 2000, pages 778 - 786, XP008077779, DOI: doi:10.1002/1521-4141(200003)30:3<778::AID-IMMU778>3.0.CO;2-I
GOLDSTEIN A.L.; BADAMCHIAN M.: "Thymosins: chemistry and biological properties in health and disease", EXPERT OPIN. BIOL. THERAPY, vol. 4, no. 4, pages 559 - 573
GOLDSTEIN, A.L.; SLATER, F.D.; WHITE, A.: "Preparation and partial purification of a thymic lymphocytopoietic factor", PROC. NATL. ACAD. SCI. USA, vol. 56, no. 3, 1966, pages 1010 - 1017
HERZENBERG, L.A.; DE ROSA, S.C.; DUBS, J.G.: "Glutathione deficiency is associated with impaired survival in HIV disease", PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 1967 - 1972
HUTCHINSON, L.M. ET AL.: "Development of a sensitive and specific enzyme-linked immunoassay for thymosin beta 15, a urinary biomarker of human prostate. cancer", CLIN. BIOCHEM., vol. 38, no. 6, 2005, pages 558 - 571
ISHITSUKA, H ET AL.: "Protective activity of thymosin against opportunistic infections in animal models", CANCER IMMUNOL. IMMUNOTHER., vol. 14, 1983, pages 145 - 150J
KONA, HAWAI ET AL.: "Thymosin alpha 1 inhibits Sendai virus replication: involvement of intracellular redox state", 6TH INTERNATIONAL EXPERT FORUM OF IMMUNOTHERAPY AND GENE THERAPY, May 1998 (1998-05-01)
LEONDIADIS, L. ET AL.: "Solid-phase synthesis of thymosin ß10 using a p-cyanotrityl resin. Chemical characterization and immunochemical control of the synthetic peptide", J. CHEM. SOC., PERKIN TRANS. 1, 1996, pages 971 - 975J
LEONDIADIS, L. ET AL.: "Solid-phase synthesis of thymosin ß10 using a p-cyanotrityl resin. Chemical characterization and immunochemical control of the synthetic peptide", J. CHEM. SOC., PERKIN TRANS., vol. 1, 1996, pages 971 - 975
MOODY, T.W. ET AL.: "Thymosin alpha1 inhibits mammary carcinogenesis in Fisher rats", PEPTIDES, vol. 23, no. 5, 2002, pages 101.1 - 1014
MOODY, T.W. ET AL.: "Thymosin alpha1 is chemopreventive for lung adenoma formation in A/J mice", CANCER LETT., vol. 155, 2000, pages 121 - 127J
PHILP D. ET AL.: "The actin binding site on thymosin ß4 promotes angiogenesis", FASEB J., vol. 17, pages 2103 - 2105, XP003009670
PICA, F. ET AL.: "High doses of thymosin alpha 1 enhance the anti-tumor efficacy of combination chemo-immunotherapy for murine B16 melanoma", ANTICANCER RES., vol. 18, 1998, pages 3571 - 3578, XP001042208
Retrieved from the Internet <URL:http://www.emdbiosciences.SharedImages/Novabiochem/03_04_innovE.pdf> *
RHO, S.B. ET AL.: "The identification of apoptosis-related residues in human thymosin ß-10 by mutational analysis and computational modelling", J. BIOL. CHEM., vol. 280, no. 40, pages 34003 - 34007
See also references of EP2019118A4
TENNANT, B.V. ET AL.: "Treatment of chronic woodchuck hepatitis virus infection with thymosin alpha- (TA1", ANTIVIRAL RES., vol. 20, no. 1, 1993, pages 1634
VASILE, E. ET AL.: "Differential expression of thymosin 13-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis", FASEB J., vol. 15, 2001, pages 458 - 466
VOELTER, W ET AL.: "Synthesis of thymosin ß4Xen and its comparison with the natural tritetracànpeptide", J. PRAKT. CHEM., vol. 341, no. 1, 1999, pages 47 - 51J
WANDS, J.R., MONTE S., RHODE ISLAND HOSPITAL (US, 30 June 2005 (2005-06-30)
WANG, S. ET AL.: "Cleavage and deprotection of peptides on MBHA-resin with hydrogen bromide", INT J. PEPT. PROT. RES., vol. 40, no. 3-4, 1992, pages 344 - 349, XP000311245
WANG, S. ET AL.: "Cleavage and deprotection of peptides on MBHA-resin with hydrogen bromide", INT.J.PEPT. PROT. RES., vol. 40, no. 3-4, 1992, pages 344 - 349, XP000311245
WHITE, P. ET AL.: "Expediting the Fmoc solid phase synthesis of long peptides through the application of dimethyloxazolidine dipeptides", J. PEPTIDE SCI., vol. 10, 2004, pages 18 - 26
WÖHR, T. ET AL.: "Pseudoprolines as a solubilizing, structure-disrupting protection technique in peptide synthesis", J. AM. CHEM. SOC., vol. 118, 1996, pages 9218 - 9227, XP002447320, DOI: doi:10.1021/ja961509q
WÖHR, T. ET AL.: "Pseudoprolines as a solubilizing, structure-disrupting protection technique in peptide synthesis", J. AM. CHEM. SOC., vol. 118, 1996, pages 9218 - 9227J
YIALOUIRIS, P.P. ET AL.: "The complete sequences of trout (Salmo gairdneri) thymosin ß11 and its homologue thymosin ß12", BIOCHEM.J., vol. 283, 1992, pages 385 - 389
YU, F.X ET AL.: "Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins", J. BIOL. CHEM., vol. 268, no. 1, 1993, pages 502 - 509
ZIKOS, C. ET AL.: "Comparative evaluation of four trityl-type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis", J. PEPT. SCI, vol. 9, no. 7, 2003, pages 419 - 429, XP009058375, DOI: doi:10.1002/psc.454

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104558149A (zh) * 2015-01-22 2015-04-29 苏州天马医药集团天吉生物制药有限公司 胸腺素α1的固相片段合成方法
CN104558149B (zh) * 2015-01-22 2018-10-26 苏州天马医药集团天吉生物制药有限公司 胸腺素α1的固相片段合成方法

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ES2288118B1 (es) 2008-11-01
EP2019118B1 (fr) 2012-11-07
ES2399248T3 (es) 2013-03-27
DK2019118T3 (da) 2013-02-04
US20110166071A1 (en) 2011-07-07
US20130172529A1 (en) 2013-07-04
AR061097A1 (es) 2008-08-06
CN101484467B (zh) 2014-01-08

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